Assessment of bioremediation potential of Calotropis procera and Nerium oleander for sustainable management of vehicular released metals in roadside soils

Land transportation is a major source of heavy metal contamination along the roadside, posing significant risks to human health through inhalation, oral ingestion, and dermal contact. Therefore, this study has been designed to determine the concentrations of vehicular released heavy metals (Cd, Pb, Ni, and Cu) in roadside soil and leaves of two commonly growing native plant species (Calotropis procera and Nerium oleander).Two busy roads i.e., Lahore-Okara road (N-5) and Okara-Faisalabad roads (OFR) in Punjab, Pakistan, were selected for the study. The data were collected from five sites along each road during four seasons. Control samples were collected ~ 50 m away from road. The metal content i.e. lead (Pb), cadmium (Cd) nickel (Ni) and copper (Cu) were determined in the plant leaves and soil by using Atomic Absorption Spectrophotometer (AAS). Significantly high amount of all studied heavy metals were observed in soil and plant leaves along both roads in contrast to control ones. The mean concentration of metals in soil ranged as Cd (2.20–6.83 mg/kg), Pb (4.53–15.29 mg/kg), Ni (29.78–101.26 mg/kg), and Cu (61.68–138.46 mg/kg) and in plant leaves Cd (0.093–0.53 mg/kg), Pb (4.31–16.34 mg/kg), Ni (4.13–16.34 mg/kg) and Cu (2.98–32.74 mg/kg). Among roads, higher metal contamination was noted along N-5 road. Significant temporal variations were also noted in metal contamination along both roads. The order of metal contamination in soil and plant leaves in different seasons was summer > autumn > spring > winter. Furthermore, the metal accumulation potential of Calotropis procera was higher than that of Nerium oleander. Therefore, for sustainable management of metal contamination, the plantation of Calotropis procera is recommended along roadsides.


Study area
To assess the concentration of traffic-related metals (Cd, Pb, Ni, and Cu,) in roadside soil and bioremediation potential of plants, two busy roads i.e., Lahore-Okara road (N-5) and Okara-Faisalabad roads (OFR) in the Punjab Pakistan, were selected as study area (Fig. 1).Both roads vary in traffic density, vehicle type, and age of roads.The National Highway (N-5) was constructed in 1913 to connect Torkham to Karachi.A very busy section of this highway, Lahore-Okara road (129 km) was selected for study.Five sites (Chung, Manga Mandi, Bhai Phero, Pattoki, and Renala Khurd) were selected.The Okara-Faisalabad road (OFR) is a newly built road with less traffic volume than N-5.Five sites along this road, Satghara More, Bangla Gogera, Tandaliawala, Sataiana, and Khanuana, were selected randomly for the study.

Sample collection and heavy metal analysis
The case study was conducted in four seasons (summer, autumn, winter, spring).The soil samples and leaves of two commonly growing plant species (Calotropis procera A., and Nerium oleander L.) were collected from all sites (0-1 m from road edge) of both roads.Control soil and plant leaf samples were collected from 100 m away from road.Five plants of each species from every site were collected during four seasons.Five soil samples were collected from 0-1 m from road edge (0-10 cm deep) from each site along both roads.Vehicular soot (Cars, trucks, and buses) and fuel (diesel, petrol) from various fuel stations along both roads were also analyzed to determine metal contents.
The soil particles on collected leaf samples were removed with deionized distilled water for acid digestion.After drying at 65 °C in a hot-air oven, all samples were milled to powder using Wiley Mill.While, soil samples were first put through a sieve (2-mm) and then dried at 65 °C in an oven for 72 h.Then, using nitric acid and hydrogen peroxide, all air-dried samples (plant leaves and soil) were processed on a hot-block digester by following USEPA, 3050B method for analysis of metals 17 .The methodology of 18 was followed for soot digestion, and 19 for petrol and diesel digestion.The Cd, Ni, Pb, and Cu contents in all digested samples were analyzed using an atomic absorption spectrophotometer (AAS).For accuracy and precision in analysis, reference materials with accuracy = 100 ± 20%, reagent blanks, and internal standard solutions were used.Traffic density (average daily traffic/season) was also recorded at all study sites (Table 1).For statistical analyses, analysis of variance was performed with the program COSTAT (Cohort Statistical Software 2003, Monterey, California, USA).Means were compared with LSD test (α = 0.05) to differentiate between different sites, plants, and seasons for metal contamination.

Ethical approval
It has been confirmed that the experimental data collection complied with relevant institutional, national, and international guidelines and legislation with appropriate permissions from authorities of the Department of Horticulture, University of the Punjab, Lahore, Lahore 54300, Pakistan.This research did not contain any studies involving animal or human participants, nor did it occur in any private or protected areas.No specific permissions were required for corresponding locations.

Heavy metal content in roadside soil
The mean concentrations of vehicular-released metals (mg/kg) in soils are given in Table 2. Metal (Cd, Pb, Ni, and Cu) content in the soil of all sites along both roads varied significantly.All studied metals were higher in roadside soils than in control site soil.In the present case study, metal content in roadside soil ranged between 2.20 and 6.83 mg/kg Cd, 4.56-15.29 mg/kg Pb, 31.58-101.26mg/kg Ni and 31.58-101.26Cu along N-5.Along Okara-Faisalabad road, the Cd, Pb, Ni, and Cu contents were found as 2.73-6.37,4.53-14.28,29.78-95.89,and 62.85-132.10mg/kg, respectively.Among sites, Chung was a highly contaminated site along N-5; along OFR, metal contamination was highest at the Tandliawala site.The comparison of roads showed higher metal contamination along N-5 road.Among seasons, the highest metal contamination in roadside soil was recorded in summer and the least in winter.

Bioremediation potential of roadside plants
The amount of metals (Cd, Pb, Ni, and Cu) in roadside plant leaves was considerably higher than the control site plants during all seasons.Among sites, the maximum amount of metal in plant leaves was observed at Chung site along N-5 and Tandaliawala site along OFR (Fig. 2).The mean metal uptake in Calotropis procera was higher than Nerium oleander (Fig. 3).Among seasons, plant metal content was highest in the summer season (Fig. 4).

Metal content in fuel and soot
High amounts of Cd, Pb, Ni, and Cu were noted in diesel, petrol, and used motor oil (Table 3).The categorization of metal content in fuel was Cd < Pb < Ni < Cu.However, the metal contents in used motor oil were highest.High metal content was also found in the soot of various vehicles, though truck soot has the highest amount of Cd, Pb, Ni, and Cu i.e., 0.97, 8.55, 12.78, and 21.51 mg/kg, respectively.

Heavy metal content in roadside soil
The mean concentrations of vehicular-released metals (mg/kg) in soils have been presented in Table 2. Significant differences existed in metal (Cd, Pb, Ni and Cu) content in the soil of all sites along both roads.All studied metals were higher in roadside soils than in control site soil.Among sites, all the metals were higher in concentration at Chung site along N-5, and along OFR it was highest at Tandliawala site.Among seasons, the highest metal contamination in roadside soil was recorded in summer and the least in winter.The roads with high traffic volume www.nature.com/scientificreports/and speedy automobiles incorporate many metals into the environment that sooner or later get deposited on the roadside soil 20 .During this study, high metal contents were found along N-5 as compared to ORF.Many earlier studies also found higher metal contamination in roadside soils 15,[21][22][23] .Cadmium (Cd) in roadside environment come from burning fuel, corrosion of radiators and batteries, and wearing old tires 24 .In the present case study, Cd content in roadside soil ranged between 2.20 and 6.83 mg kg −1 along N-5 and 2.73-6.37 mg kg −1 along Okara-Faisalabad road.Many earlier researchers also found higher Cd content in roadside soil 15,25,26 .
Lead (Pb) is a most important autoexhaust pollutant 27 .Regardless of the ban on leaded fuel, it is still found in petrol.In the current study, the content of Pb in roadside soil was significantly higher in contrast to control Table 2. Metal contents (mg/kg) in the roadside soil along N-5 and OFR during different seasons (mean ± SD).N-5 Lahore to Okara road, OFR Okara-Faisalabad road.Letters 'abcdefg' represent significant difference among sites.Same letters on different values signify non-significant differences (Fisher's LSD 0.05).www.nature.com/scientificreports/site soil.These results conform with many earlier studies 26,[28][29][30][31] .The mean Pb content in roadside soil along N-5 ranged between 4.56 and 15.29 mg/kg, and along Okara-Faisalabad road it ranged 4.53-14.28mg/kg.These results showed that the vehicles are the key source of Pb contamination in roadside soil 32 .Nickel (Ni) concentration in roadside soil was significantly higher than control site soil.The mean Ni content in roadside soil along N-5 ranged between 31.58 and 101.26 mg/kg, and along Okara-Faisalabad road it ranged 29.78-95.89mg/kg.Many earlier researchers also reported higher levels of metals in roadside soil 33,34 .The Ni in roadside soil might have come from engine oil, fuel, tire, and brake wear 12 and corrosion of nickel alloy bearings, valves, and shafts 35 .

Metals
The amount of Cu in roadside soil (61.68-138.46mg/kg along N-5 and 62.85-132.10mg/kg along Okara-Faisalabad road) was significantly higher than in control site soil.These results parallel many former studies [36][37][38] .This showed that the traffic is one of the major sources of Cu contamination in soil 39 .Automobiles released Cu into the surrounding environment from fuel burning, battery corrosion 38 and brakes and tire wear 40 .The metal content (Cd, Pb, Ni and Cu) found during present study was higher than the permissible limit of these metals in the soil set by 41 (Table 4).www.nature.com/scientificreports/

Heavy metal content in roadside plants
Plants that grow on heavy metal-polluted soil uptake those metals along with other essential minerals and accumulate them in different body parts 16 .The metal content (Cd, Pb, Ni, and Cu) found along N-5 and OFR were above the permissible limit of these metals in the plants set by WHO (Table 4).Compared to common plant species, metal accumulating plants typically exhibit unique and distinct compartmentation and build different complexes 44 .Metals like cadmium induces phytochelatins (PCs) formation in plants; however, it is bound by weak oxygen ligands as a substitute for strong sulfur ligands in metal hyperaccumulators 45 .This is distinctive of many metal accumulating plants 44 .In plants, the accumulation of different metals is also influenced by environmental factors and plant genotypes 46 .So, it is different in different plants, even under the same environments 47 .Plant physiological mechanisms play an important role in metal accumulating plants to alleviate metal toxicity.Cell binds the metal with the cell wall and immobilizes it, thus alleviating metal toxicity in plants 48 .Furthermore, metal that enters the cell bonds to various organic ligands to produce stable chelates that are subsequently taken up by the vacuole 49 .In the current study, the metal accumulation potential of Calotropis procera was significantly higher than that Nerium oleander.So, both plants could be used to monitor metal contamination, while Calotropis procera could be a better choice to remediate the metal-contaminated soil.

Spatial variations in heavy metal contents in roadside soil and plant leaves
The difference in metal contamination at different sites is directly related to the traffic density of that particular site.Higher metal content in soil and plant leaves at Chung and Tandaliawala was due to high traffic flow at these sites 11 .Furthermore, Pattoki site along N-5 was the least contaminated site among all other sites.This is because of high vegetation protection along this site, as many plants can naturally ameliorate metal-contaminated areas 50 .The higher concentrations of metals along N-5 are attributed to higher traffic volume along this road.Besides traffic density, several other factors i.e., road structure, type of vehicles, fuel, and age of roads 51 , also contribute to metal contamination near roads.The national highway (N-5) is a very old concrete road, and older roads retain significant amount of deposited metals in nearby soil 26 .Furthermore, concrete roads cause more metal contamination than asphalt roads 52 .The speed of vehicles along N-5 remains very high compared to OFR, which causes more tire wear and tear, resulting in high metal content in the surrounding environment 26 .The reason for less metal content along OFR is that it is newly constructed and less busy than N-5 road.However, high metal  www.nature.com/scientificreports/contents were recorded at the Tandaliawala site, which act as junctions/temporary bus stops for passenger vans.This is supported by the results of 53 , who found high metal content at traffic junctions and crossroads.

Seasonal variation in heavy metal content in roadside soil and plant leaves
Metal contamination in roadside soil and plant leaves was higher in the summer and lowest in winter.The high metal contamination in summer might be due to high traffic density and more rubber abrasion at high temperatures (average day temperature is 43.5 °C).Furthermore, metals are more bioavailable at high temperatures, and plant accumulation is higher in plants during summer 3 .Metals percolate deep into the soil due to heavy precipitation during late summer 54,56,57 also observed seasonal variation in metal contamination.

Metal content in fuel and soot
Despite the ban on leaded fuel, Pb was still found in petrol, diesel, and used motors during the current investigation.Lead-containing petrol is always considered a major cause of Pb pollution.Metal content in automobiles' fuel found during the present study was greater than those reported by 55 .They noted 0.04 μg/g Cd, 4.50 μg/g Pb, 0.22 μg/g Ni, and 7.00 μg/g Cu in used motor oil, while Cd and Cu were not detectable in unused oil and Pb was lower in concentration (2.00 μg/g) in unused oil.This indicates that metals are released from automobiles during different process 3,35 also reported metal content in soot of vehicles.The higher amount of metals in fuel and soot of vehicles proved our findings that the vehicles are the main contributors of metal pollutants in the environment.

Conclusion
Heavy metal (Cd, Pb, Ni, and Cu) concentrations in roadside soil and plant leaves were much higher than the standard permissible limits.They showed a strong positive connection with traffic density.This demonstrates that automobiles are the primary source of heavy metal contamination in the environment near roadsides.
Residents living close to roads and agriculture may suffer as a result.For control to be effective, heavy metal contamination must be regularly monitored.The current study focuses on employing native plant species to reduce soil contamination with Cd, Pb, Ni, and Cu as a sustainable management strategy.Calotropis procera is a useful metal accumulator.Therefore, it could be used to reclaim the heavy metal-contaminated soils.Though this study offers valuable insights, it is imperative to recognize its limits.The study locations chosen are urban areas, and more research in other locations and climates could enhance the conclusions' generalizability.Furthermore, long-term monitoring and assessing their performance would provide a more thorough knowledge of the chosen tree species' potential as useful instruments for improving environmental quality.Altogether, our research highlights the critical role that native vegetation plays in reducing environmental pollution and offers insightful advice to environmentalists, legislators, and urban planners on how to create more sustainable and healthy urban settings.We can get one step closer to creating more resilient and environmentally friendly cities for the benefit of present and future generations by incorporating suitable tree species into urban landscapes.

Figure 2 .Figure 3 .
Figure 2. Metal content in plants leaves collected from various sites along N-5 and OFR during different seasons where, (a) Cd contents in plant leaves, (b) Pb contents in plant leaves, (c) Ni contents in plant leaves, (d) Cu contents in plant leaves.N-5 Lahore to Okara road, OFR Okara-Faisalabad road.

Figure 4 .
Figure 4. Metal uptake in plants leaves collected from various sites along N-5 and OFR during different seasons where, (a) Cd contents in plant leaves, (b) Pb contents in plant leaves, (c) Ni contents in plant leaves, (d) Cu contents in plant leaves.N-5 Lahore to Okara road, OFR Okara-Faisalabad road.

Table 1 .
Average daily traffic (number of vehicles/day) at studied sites.N-5 Lahore to Okara road, OFR Okara-Faisalabad road.

Table 3 .
Metal content in fuel (petrol and diesel), used motor oil and soot (mean ± S.D.).

Table 4 .
Permissible limits of heavy metal contents (mg/kg) in soil and plants.